1000 µl PreAMP mix for a user-defined set of loci — enough for 250 reactions

Principle

For successful somatic mutation detection, experimental methods must detect the DNA molecules with mutations of interest in samples that may contain a vast excess of DNA molecules with the wildtype sequence at the same locus. Based off of this principle, there are two factors that affect these experiments: the inherent percentage of mutant DNA in a sample, and the sensitivity of the method. In the case of cancer, a specific mutation may be found in a small percentage of the tumor, which is a characteristic of tumor heterogeneity. Additionally, different types of samples and sample sources affect this ratio of mutant to wildtype DNA as well as the number of DNA molecules that can be assayed. A method's sensitivity directly reflects how few mutant DNA molecules can be detected within a given amount of wildtype DNA.

Sample types common in cancer research, including formalin-fixed paraffin embedded (FFPE) samples, serum samples, fine needle biopsies, and sorted cells frequently contain only small amounts of amplifiable DNA. Genomic DNA in FFPE samples is fragmented, for example, while DNA in serum samples is heavily diluted. In the case of fine needle biopsies or sorted cells, these samples have small numbers of cells that will yield limited amounts of genomic DNA. Amplifying genomic DNA enables more accurate detection of the mutations in DNA from these sample types.

The qBiomarker PreAMP system uses nested PCR preamplification to improve specificity and decrease false positives. In a 12-cycle PCR reaction, the loci of interest are amplified and are ready to be analyzed with qBiomarker Somatic Mutation PCR Assays and Arrays. The Custom qBiomarker Array PreAMP Mix is a mix of primers for a user-defined set of loci. In combination with the qBiomarker PreAMP Synthesis Kit, the mix amplifies this set of loci for analysis with the Custom qBiomarker Somatic Mutation PCR Array.

Procedure

DNA is first isolated from FFPE, serum, or small samples using the appropriate QIAamp or DNeasy kit. The qBiomarker PreAMP Synthesis Kit and Custom qBiomarker PreAMP Mix are then used to preamplify the DNA using locus-specific primers. A Custom qBiomarker Somatic Mutation Array is then used for mutation detection, and CT values are uploaded into the GeneGlobe Data Analysis tool for data analysis and mutation calls.